Identification of domains on the extrinsic 23 kDa protein possibly involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II Akihiko Tohri1,2, Nao
Trang 1Identification of domains on the extrinsic 23 kDa protein possibly involved in electrostatic interaction with the extrinsic 33 kDa protein
in spinach photosystem II
Akihiko Tohri1,2, Naoshi Dohmae3, Takehiro Suzuki1, Hisataka Ohta1,4, Yasunori Inoue2,4and Isao Enami1 1
Department of Biology, Faculty of Science, Tokyo University of Science, Kagurazaka, Shinjuku-ku, Tokyo, Japan;
2
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Yamazaki,
Noda, Chiba, Japan; 3 Division of Biochemical Characterization, the Institute of Physical and Chemical Research (RIKEN), Hirosawa, Wako, Saitama, Japan; 4 Tissue Engineering Research Center, Tokyo University of Science, Yamazaki, Noda, Chiba, Japan
To elucidate the domains on the extrinsic 23 kDa protein
involved in electrostatic interaction with the extrinsic 33 kDa
protein in spinach photosystem II, we modified amino or
carboxyl groups ofthe 23 kDa protein to uncharged methyl
ester groups with N-succinimidyl propionate or glycine
methyl ester in the presence ofa water-soluble
carbodi-imide, respectively The N-succinimidyl propionate-modified
23 kDa protein did not bind to the 33 kDa protein
associ-ated with PSII membranes, whereas the glycine methyl
ester-modified 23 kDa protein completely bound This indicates
that positive charges on the 23 kDa protein are important for
electrostatic interaction with the 33 kDa protein associated
with the PSII membranes Mapping ofthe N-succinimidyl
propionate-modified sites ofthe 23 kDa protein was
per-formed using Staphylococcus V8 protease digestion ofthe
modified protein followed by determination of the mass of
the resultant peptide fragments with MALDI-TOF MS The results showed that six domains (Lys11–Lys14, Lys27– Lys38, Lys40, Lys90–Lys96, Lys143–Lys152, Lys166– Lys174) were modified with N-succinimidyl propionate In these domains, Lys11, Lys13, Lys33, Lys38, Lys143, Lys166, Lys170 and Lys174 were wholly conserved in the 23 kDa protein from 12 species of higher plants These positively charged lysyl residues on the 23 kDa protein may be involved
in electrostatic interactions with the negatively charged carboxyl groups on the 33 kDa protein, the latter has been suggested to be important for the 23 kDa binding [Bricker, T.M & Frankel, L.K (2003) Biochemistry 42, 2056–2061] Keywords: extrinsic 23 kDa protein; extrinsic 33 kDa pro-tein; electrostatic interaction; chemical modification; oxygen evolution
Photosystem II (PSII) catalyzes the light-driven oxidation
ofwater with concomitant reduction ofplastoquinone to
plastoquinol This multisubunit protein-pigment complex
contains a number ofintrinsic proteins and 3–4 extrinsic
proteins associated with the lumenal side ofPS II The three
extrinsic proteins of33, 23 and 17 kDa associate with higher
plant and green algal PSII [1] Their binding properties,
however, are different between higher plant and green algal
PSII In higher plant PSII, the 33 kDa protein associates
directly with PSII, but the 23 kDa protein cannot directly
bind to PSII and associates with PSII only through its
interaction with the 33 kDa protein, and the 17 kDa protein
functionally associates with PSII only through its inter-action with both the 33 and 23 kDa proteins [2] The 23 and
17 kDa proteins are easily released from higher plant PSII
by washing with 1M NaCl, indicating that the 23 kDa protein electrostatically binds to the 33 kDa protein [3], and the 17 kDa protein interacts electrostatically with both the
33 and 23 kDa proteins In contrast, the green algal 23 and
17 kDa proteins can bind directly to PSII independent of the presence or absence ofother extrinsic proteins [4] On the other hand, cyanobacterial PSII contains three extrinsic proteins of33 and 12 kDa, and cytochrome c550 [5], whereas, red algal PSII contains four extrinsic proteins of
33, 20 and 12 kDa, and cytochrome c550 [6,7]
The extrinsic proteins play important roles for maximal rates ofoxygen evolution under physiological ionic condi-tions [1] The 33 kDa protein is needed to maintain the functional conformation of the Mn cluster [8,9] Shutova
et al found that titration of the 33 kDa protein against pH
in solution exhibited a striking hysteresis [10], and proposed that the protein is not only required for stabilizing the Mn-cluster but also important for proton transport to occur appropriately, accompanying oxygen evolution [11] The functions of the 23 and 17 kDa proteins are closely related with the unique requirement ofCa2+and Cl–for oxygen evolution; the 23 kDa protein mitigates the demand for
Ca2+ while the 17 kDa protein does for Cl– [8,12–14]
Correspondence to I Enami, Department ofBiology, Faculty of
Science, Tokyo University ofScience, Kagurazaka 1-3, Shinjuku-ku,
Tokyo 162-8601, Japan Tel.: + 81 4 7124 1501 (ext 5022),
E-mail: enami@rs.noda.tus.ac.jp
Abbreviations: CBB, Coomasie brilliant blue; Chl, chlorophyll; CHC,
a-cyano-4-hydroxycinnamic acid; DHB, 2,5-dihydroxybenzoic acid;
EDC, 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide; GME,
glycine methyl ester; MBT, 2-mercaptobenzothiazole;
NHS, N-hydroxysuccinimido; NSP, N-succinimidyl propionate;
PSII, photosystem II.
(Received 28 October 2003, revised 9 January 2004,
accepted 16 January 2004)
Trang 2The extrinsic proteins of12 kDa and cytochrome c550
in cyanobacterial and red algal PSIIs have a similar
func-tion to that ofthe 23 and 17 kDa proteins in higher
plant PSII [7,15–17]
Recently, Zouni et al [18] and Kamiya and Shen [19]
published the crystal structural analysis ofthermophilic
cyanobacterial PSII These studies have provided important
insights into the organization ofnumerous subunits of
cyanobacterial PSII The 33 kDa protein and cytochrome
c550 appear to interact with the large extrinsic loop E of
CP47 and with the large extrinsic loop E ofCP43,
respect-ively The 12 kDa protein appears to interact with both the
33 kDa protein and cytochrome c550 [19] These studies
provided, however, few insights into the structural
organ-ization ofthe 23 and 17 kDa proteins in higher plant PSII
Three-dimensional crystals from higher plant PSII uniformly
diffract poorly [20] and two-dimensional crystals examined
by electron diffraction have been performed at low
resolutions with PSII from higher plants devoid of an
oxygen-evolving complex [21,22] In cross-reconstitution
experiments, the 23 and 17 kDa proteins bound to
cyano-bacterial and red algal PSII only through non-specific
interactions [16] The CaCl2-washed spinach PS II
mem-branes which had been reconstituted with either
cyanobac-terial or red algal 33 kDa protein, could only partially rebind
spinach 23 kDa protein but could not bind spinach 17 kDa
protein [23] These data indicate that there are structural
determinants present on the spinach 33 kDa protein that are
required for the efficient binding of the 23 and 17 kDa
proteins and that are absent in cyanobacterial and red algal
proteins [24]
The organization among the three extrinsic proteins
in spinach PSII has been examined by cross-linking
experiments Cross-linking experiments performed with
homobifunctional cross-linkers (6–14 A˚ span) indicated
that the 33 kDa protein is within a distance of11 A˚ of
the 23 kDa protein and that the 23 kDa protein is within
11 A˚ ofthe 17 kDa protein [25] This indicates that these
three extrinsic proteins must be in close proximity
Cross-linking experiments also showed that the 33 kDa protein is
associated with or in close proximity to CP47 [25–28],
D1 and D2 [29], a large subunit ofcytochrome b559 [30]
and PsbI [30] The 33 kDa protein was shown to be also
associated with CP43 by comparing the peptide mappings
ofthe trypsin-digested products ofNaCl-washed and
CaCl2-washed PSII membranes [31] Thus, the 33 kDa
protein is associated with or in close proximity to essentially
all ofthe major intrinsic proteins in higher plant PSII
Chemical modification is a useful method to elucidate
which positive or negative charges on the extrinsic proteins
are responsible for electrostatic interaction with the other
extrinsic proteins and/or the intrinsic proteins [32,33] We
have reported that the N-succinimidyl propionate
(NSP)-modified 33 kDa protein, ofwhich the positively charged
amino groups are modified to uncharged methyl ester groups
[33], cannot rebind to spinach PSII, whereas the glycine
methyl ester (GME)-modified protein, ofwhich the
negat-ively charged carboxyl groups are modified to uncharged
methyl ester groups [32], can rebind and reactivate the
oxygen evolution [34] These results indicate that positive
charges on the 33 kDa protein are important for its
electrostatic interaction with PSII intrinsic proteins, whereas
negative charges on the protein do not contribute to such interaction The domains ofthe 33 kDa protein possibly involved in electrostatic interaction with PSII intrinsic proteins were also determined to be Lys4, Lys20, Lys66– Lys76, Lys101, Lys105, Lys130, Lys159, Lys186 and Lys230–Lys236 by a combination ofV8 protease digestion and MALDI-TOF MS ofNSP or 2,4,6-trinitrobenzene sulfonic acid-modified 33 kDa protein [34], or NHS-biotin modified one [35] Furthermore, we showed that a similar number ofcarboxyl groups on the 33 kDa protein were modified with GME in both the protein in solution and bound to PSII [34] This suggests that most ofthe carboxyl groups on the 33 kDa protein are not located in regions interacting with PSII intrinsic proteins and exposed to the lumenal side ofPSII Thus, we hypothesized that negative charges ofcarboxyl groups on the 33 kDa protein may be involved in electrostatic interaction with the 23 and 17 kDa proteins In fact, Bricker and Frankel [24] showed recently, that spinach PS II membranes reconstituted with the 33 kDa protein, on which the negatively charged carboxyl groups were modified with GME, was defective in its ability to bind the 23 kDa protein ofPSII They hypothesized that the domains on the 33 kDa protein possibly involved in electrostatic interaction with the 23 kDa protein are Glu1, Glu32, Glu139 and/or Glu187, which are wholly conserved
in higher plants but which are poorly conserved in cyano-bacteria These facts in turn suggest that positive charges on the 23 kDa protein may be responsible for the electrostatic interaction with these negative charges on the 33 kDa protein
The binding domains ofthe 23 kDa protein, however, remain obscure Recently, Ifuku and Sato [36] reported that the binding affinity of a recombinant mutant of the 23 kDa protein, ofwhich N-terminal 19 residues were truncated, were apparently weaker than that ofthe native 23 kDa protein, and the mutant protein completely lacked the ability to retain Ca2+for oxygen evolution This suggests that the N-terminal region ofthe 23 kDa protein is important for its binding with the 33 kDa protein
In the present study, the domains on the 23 kDa protein possibly involved in electrostatic interaction with the 33 kDa protein associated with PSII membranes were examined
by chemical modification method The results showed that positive charges on the 23 kDa protein are indeed important for its interaction with the 33 kDa protein, and we have determined the domains ofpositive charges on the 23 kDa protein that are possibly involved in the interaction
Materials and methods
Preparations Oxygen-evolving PSII membranes were prepared from spinach chloroplasts with Triton X-100 as described in Berthold et al [37], with slight modifications [28] The isolated PSII membranes were suspended in medium A (40 mMMes/NaOH, pH 6.5; 0.4M sucrose; 10 mMNaCl and 5 mMMgCl2, and stored in liquid nitrogen until used The extrinsic 33 and 23 kDa proteins were extracted from the PSII membranes by 1M CaCl2 treatment, incubated with 1MCaCl2for 3 h in the dark to suppress the activity ofcopurified protease, dialyzed against 5 m Mes/NaOH,
Trang 3pH 6.5 and further against 20 mMphosphate buffer, pH 6.5
and then purified by column chromatography with a
DEAE-Sepharose CL-6B column (Pharmacia Biotech Inc.,
NJ, USA) [16,38] The concentrations ofthe 33 and 23 kDa
proteins were determined using an extinction coefficient of
16 mM )1Æcm)1at 276 nm [39] and 26 mM )1Æcm)1at 277 nm
[38], respectively
Chemical modification
For modification ofamino groups oflysyl residues and the
free amino terminus of the 23 kDa protein, the purified
protein (30 lM) was incubated in a reaction mixture
containing 20 mM phosphate buffer, pH 6.5 and
0.5–6.0 mMNSP at 25°C for 90 min The reaction mixtures
were dialyzed against 10 mM Mes/NaOH, pH 6.5 to
remove unreacted NSP Chemical modification ofcarboxyl
groups on the purified 23 kDa protein was performed in
100 mM GME, pH 6.2 containing 30 lM ofthe 23 kDa
protein and 2 mM1-ethyl-3-(3-(dimethylamino)propyl)
car-bodiimide (EDC) at 25°C for 12 h The reaction mixture
was dialyzed against 1M NaCl and 20 mM phosphate
buffer, pH 6.5 to remove unreacted and electrostatically
attached reagents, and then against 10 mM Mes/NaOH,
pH 6.5 NSP was purchased from Wako Pure Chemicals
(Tokyo, Japan), and GME and EDC were purchased from
Nacalai Tesque Chemicals (Tokyo, Japan)
Reconstitution and electrophoresis
For reconstitution, PS II membranes were washed with
2.6MUrea, 0.2M NaCl in the dark to remove the three
extrinsic proteins of33, 23 and 17 kDa [8] The resultant
PSII membranes were incubated with the 33 kDa protein
and with either the unmodified or modified 23 kDa protein
at a protein-Chl ratio of0.6 (w/w), in medium A at 0°C f or
30 min in the dark at a Chl concentration of0.5 mg mL)1
The reconstituted PSII membranes were collected by
centrifugation at 35 000 g for 10 min and then washed
once with and resuspended in medium A The reconstituted
PSII membranes were again treated with 2.6Murea, 0.2M
NaCl in the dark for 30 min and the centrifuged
super-natants were applied on SDS/PAGE to estimate the
amounts ofthe 33 kDa and 23 kDa proteins rebound by
the reconstitution
SDS/PAGE was performed with a gradient gel of 16–
22% acrylamide containing 7.5 urea [40] Samples were
solubilized with 5% lithium lauryl sulfate and 75 mM
dithiothreitol The amounts ofrebound 23 kDa protein
were determined from the integrated optical densities of the
23 kDa bands using the program NIH IMAGE (National
Institutes ofHealth, USA) after the SDS/PAGE was
scanned using a CanoScan N656U (Canon, Tokyo)
Isoelectric focusing was performed using a 5.5%
poly-acrylamide containing homogenous gel covering a pH range
of3.5–10.0 or 4.0–6.0 using 5% (v/v) ampholine
(Amer-sham Pharmacia Biotech AB, Sweden) Proteins were
stained with 0.048% CBB in 30% methanol and 10%
acetic acid
Oxygen evolution was measured with a Clark-type
oxygen electrode in 40 mMMes/NaOH, pH 6.5 and 0.4M
sucrose (medium B) at 25°C in the absence and presence of
10 mM NaCl or 5 mM CaCl2, with 0.4 mM phenyl-p-benzoquinone as the electron acceptor
Chl concentration was determined by the method of Porra et al [41]
Protease digestion The 23 kDa protein (3 nmol) modified with 0.5 or 4 mM NSP was dried and solubilized in 10 lL of 1MTris/HCl,
pH 8.5, 8Mguanidine/HCl, 1 mMEDTA and 1% dithio-threitol, and incubated at 37°C for 2 h to denature the
23 kDa protein Then, 5 lL of5% iodoacetamide was added and incubated at 37°C for 30 min to block SH groups The reaction mixtures were added to a final concentration of10% ofcold trichloroacetic acid and centrifuged, and the resulting precipitates were washed twice with acetone The final precipitates were dried and resolubilized in 20 lL of 0.1M ammonium bicarbonate After 1 lg of Staphylococcus V8 protease (ICN Biomedicals, OH, USA) was added, the
23 kDa protein was digested at 37°C, overnight and then desalted by Ziptipl-C18 (Millipore, MA, USA)
Mass spectroscopic analysis The protease-digested protein was applied directly to a MALDI-TOF MS (Reflex; Bruker Daltonics, MA, USA), with a matrix of a-cyano-4-hydroxycinnamic acid (CHC), 2-mercaptobenzothiazole (MBT) or 2,5-dihydroxybenzoic acid (DHB) The mass ofeach measured peptide fragment was assigned to the known 23 kDa protein sequence
Results
As described above, Bricker and Frankel [24] showed that negatively charged carboxyl groups on the extrinsic 33 kDa protein are important for electrostatic interaction with the extrinsic 23 kDa protein This suggests that positive charges
on the 23 kDa protein may electrostatically interact with the negative charges on the 33 kDa protein To confirm this, we modified positively charged amino groups on the 23 kDa protein to uncharged methyl ester groups with NSP Figure 1A shows the isoelectric focusing of the NSP-modified 23 kDa protein The pI value shifted toward acidic pH with increasing NSP concentration For exam-ple, the pI value downshifted from 6.8 (unmodified protein, lane 1) to 4.8–5.5 (0.5 mMNSP-modified protein, lane 2) and 4.3–4.8 (4 mM NSP-modified protein, lane 5) These changes were estimated to result from modification of1–5 amino groups in 0.5 mM NSP-modified protein and 5–10 amino groups in 4 mMNSP-modified protein to uncharged groups, as calculated using a computer pI/Mr tool [42] It should be noted here that the band ofthe modified protein appeared much broader than the unmodified protein upon isoelectric focusing, implying that the resulting protein products may be composed of proteins with different numbers of amino residues modi-fied This is similar to the results obtained by modification ofthe 33 kDa protein with NHS-biotin [35], NSP and 2,4,6-trinitrobenzen sulfonic acid [34], or GME [24]
In order to determine whether elimination ofsurface positive charges affected binding of the 23 kDa protein, the ability ofthe NSP-modified protein to rebind with the
Trang 433 kDa protein associated with PSII membranes was
examined Urea/NaCl-washed PSII membranes in which
the three extrinsic proteins of33, 23 and 17 kDa had been
removed, were reconstituted with the unmodified and
NSP-modified 23 kDa protein together with the 33 kDa protein
The reconstituted PSII membranes were again treated with
2.6 M urea plus 0.2M NaCl, and the supernatants after
centrifugation were analyzed by SDS/PAGE to determine
the amounts ofthe 33 and 23 kDa proteins rebound As
shown in Fig 2, the native 33 and 23 kDa proteins
completely rebound to urea/NaCl-washed PSII membranes
(lane 4), whereas the binding abilities ofNSP-modified
23 kDa protein decreased with increasing NSP
concentra-tion (lanes 5–9) and this ability was completely lost with
NSP treatments above 4 mM(lanes 8 and 9) This suggests
that positive charges on the 23 kDa protein are important
for electrostatic interaction with the 33 kDa protein
Table 1 shows the reactivation ofoxygen evolution by
reconstitution ofthe 23 kDa protein modified with various
concentrations ofNSP When the 33 kDa protein was
reconstituted with urea/NaCl-washed PSII membranes in
which no oxygen evolution was detected even in the
presence ofCaCl2, the oxygen evolution was reactivated
to 0, 96 and 252 lmol O2Æmg chl)1Æh)1in the absence and presence of10 mMNaCl and 5 mMCaCl2, respectively The activity further recovered to 142 and 243 lmol O2Æmg chl)1Æh)1in the absence and presence of10 mMNaCl by additional reconstitution ofthe unmodified 23 kDa protein, though little effects were detected on the activity in the presence of5 mMCaCl2 by the additional reconstitution
Fig 2 Reconstitution of the unmodified, NSP- or GME-modified
23 kDa protein together with the 33 kDa protein with urea/NaCl-washed PSII membranes Urea/NaCl-urea/NaCl-washed PSII membranes were reconstituted with the unmodified, NSP- or GME-modified 23 kDa protein together with the 33 kDa protein The reconstituted PSII membranes were again treated with 2.6 M urea, 0.2 M NaCl and their centrifuged supernatants were analyzed by SDS/PAGE to determine the amounts ofthe 33 and 23 kDa proteins rebound after reconstitu-tion Lane 1, unwashed PSII-membranes; lane 2, urea/NaCl-washed PSII membranes; lane 3, urea/NaCl–washed PSII membranes recon-stituted with the 33 kDa protein; lane 4, urea/NaCl–washed PSII reconstituted with the 33 kDa protein and unmodified 23 kDa protein; lanes 5–9, urea/NaCl–washed PSII membranes reconstituted with the
33 kDa protein and the 23 kDa protein modified by 0.5 m M NSP (lane 5), 1 m M NSP (lane 6), 2 m M NSP (lane 7), 4 m M NSP (lane 8), and 6 m M NSP (lane 9); lane 10, urea/NaCl–washed PSII membranes reconstituted with the 33 kDa protein and the GME-modified 23 kDa protein.
Fig 1 Isoelectric focusing of the NSP- (A) or GME- (B) modified
23 kDa protein (A) Lane 1, unmodified 23 kDa protein; lanes 2–6, the
23 kDa protein modified by NSP at concentrations of0.5 m M (lane 2),
1 m M (lane 3), 2 m M (lane 4), 4 m M (lane 5), 6 m M (lane 6) (B) Lane 1,
unmodified 23 kDa protein; lane 2, the 23 kDa protein modified with
100 m M GME in the presence of2 m M EDC at 25 °C f or 12 h.
Table 1 Reactivation of oxygen evolution by reconstitution of the NSP- or GME-modified 23 kDa protein to urea/NaCl-washed PSII membranes reconstituted with the 33 kDa protein Values shown are the averages ofthree measurements 23, 23 kDa protein; 33, 33 kDa protein.
PS II membrane treatment
Oxygen evolution [lmol O 2 Æ(mg chl))1Æh)1] –Ion (%) +10 mM NaCl (%) +5 mM CaCl 2 (%) Control PSII membranes 523 ± 26 (100) 525 ± 17 (100) 535 ± 18 (100)
+ 33 + 0.5 mM NSP-modified 23 25 ± 5 (5) 120 ± 9 (23) 265 ± 11 (50) + 33 + 1.0 mM NSP-modified 23 13 ± 3 (2) 110 ± 7 (21) 267 ± 12 (50) + 33 + 2.0 mM NSP-modified 23 7 ± 2 (1) 103 ± 7 (20) 260 ± 10 (49) + 33 + 4.0 mM NSP-modified 23 0 ± 0 (0) 95 ± 5 (18) 263 ± 12 (49) + 33 + 6.0 mM NSP-modified 23 0 ± 0 (0) 94 ± 6 (18) 253 ± 10 (47) + 33 + GME-modified 23 140 ± 9 (27) 250 ± 9 (48) 252 ± 12 (47)
Trang 5reconstituted together with the 33 kDa protein, their
reactivations in the absence and presence of10 mMNaCl
decreased with increasing NSP concentrations, and no
reactivation effects were observed in PSII membranes
reconstituted with the 23 kDa protein modified with NSP
above 4 mM
Figure 3 shows the correlation between the amounts of
rebound 23 kDa protein (Fig 2) and reactivation ofoxygen
evolution in the absence (open circles) and presence (closed
circles) of10 mMNaCl (Table 1) Their good correlation
indicates that loss ofthe reactivating capability ofthe
NSP-modified 23 kDa protein was caused directly by loss oftheir
rebinding, which in turn suggests that the modified protein,
when rebound, are fully functional and that there is
apparently no nonspecific binding ofthe modified protein
In contrast to the NSP-modified 23 kDa protein, the
GME-modified 23 kDa protein retained its capabilities to
rebind with the 33 kDa protein associated with PSII and to
reactivate the oxygen evolution Figure 1B shows that the
pI values were upshifted from 6.8 of unmodified protein
(lane 1) to 9.2 (lane 2) by modification ofcarboxyl groups
with GME in the presence ofEDC This change was
estimated to result from modification of around three
negatively charged carboxyl groups to uncharged groups, as
calculated using a computer pI/Mr tool The
GME-modified 23 kDa protein completely rebound to the
33 kDa protein associated with PSII membranes (Fig 2,
lane 10) and its rebinding reactivated the oxygen evolution
to extents comparable with the rebinding ofthe unmodified
23 kDa protein (Table 1) These results clearly indicate that
surface negative charges on the 23 kDa protein do not
participate in its functional binding with the 33 kDa protein
associated with PSII membranes
Next, we attempted to identify the lysyl residues on the
23 kDa protein modified with NSP Both ofthe modified
23 kDa proteins treated with 0.5 mMNSP and 4 mMNSP
whose binding abilities were lost by about 82% and 100%,
were denatured with urea and digested with Staphylococcus V8 protease followed by determination of the mass of the resultant peptide fragments with mass spectroscopy Whether a peptide fragment can be detected by the MALDI-TOF MS depends in some cases on the matrix employed, three different matrices were used: They were, a-cyano-4-hydroxycinnamic acid (CHC), 2-mercapto-benzothiazole (MBT) and 2,5-dihydroxybenzoic acid (DHB) This led to a more complete identification of the peptide fragments resulting from the V8 protease digestion ofthe modified 23 kDa protein The results were shown in Table 2 (the 23 kDa protein modified with 0.5 mM NSP) and Table 3 (the 23 kDa protein modified with 4 mM NSP) Peptide fragments yielded could be assigned to the known amino acid sequence within a 0.01% mass error, as shown in Tables 2 and 3 Modifi-cation ofthe amino group with each NSP molecule results
in an addition ofan N-propionyl group, which corres-ponds to an increase of56.0 Da in the molecular mass In the 23 kDa protein modified with 0.5 mM NSP, there were 31 peptides identified ranging in mass from 703.32 to 2840.49 Da (Table 2) Ofthese peptides, eight lysyl residues were identified to be modified with NSP, two Lys between Lys11 and Lys14; one Lys among Lys27 and Lys38; one Lys at Lys40; one Lys at Lys90 or Lys96; one Lys at Lys143 or Lys152; two Lys between Lys166 and Lys174 (Table 2) These modified lysyl residues were arranged in the amino acid sequence ofthe 23 kDa protein as shown in Fig 4 This indicates that eight lysyl residues modified with 0.5 mM NSP are located in six domains, namely Lys11–Lys14, Lys27–Lys38, Lys40, Lys90–Lys96, Lys143–Lys152, Lys166–Lys174 In the
23 kDa protein modified with 4 mM NSP, 32 peptides ranging in mass from 703.33 to 2760.30 Da were identi-fied Ofthese peptides, 11 lysyl residues were identified to
be modified with NSP, which were two Lys between Lys11 to Lys14; two Lys between Lys27 and Lys38; one Lys at Lys40; one Lys at Lys68 or Lys69; one Lys at Lys90 or Lys96; one Lys at Lys143 or Lys152 and three Lys between Lys166 and Lys174 (Table 3) Ten residues
in these modified Lys were found in the six domains that were identified to be modified with 0.5 mM NSP, as shown in Fig 4 Only one domain ofLys68–Lys69 was modified uniquely with 4 mM NSP in addition to the six domains
Discussion
The present results clearly demonstrated that modification ofamino groups on the 23 kDa protein with NSP significantly affected its rebinding ability and thus the reactivating capability ofoxygen evolution In contrast, modification ofcarboxyl groups on the protein with GME in the presence ofEDC did not affect the rebinding and reactivation capabilities We thus conclude that the positive charges, but not the negative charges, on the
23 kDa protein, are important for its interaction with PSII and in particular, the 33 kDa protein associated with PSII
The 23 kDa protein from spinach is composed of 186 amino acid residues including 14 Asp, 10 Glu, 20 Lys, and 3 Arg [43] In the present study, around three carboxyl groups
Fig 3 Relationship between the amounts of NSP-modified 23 kDa
protein rebound and oxygen evolution restored s, oxygen evolution
in the absence ofNaCl; d, oxygen evolution in the presence of
10 m M NaCl.
Trang 6were estimated to be modified with GME within the total of
24 carboxyl groups ofAsp + Glu in the 23 kDa protein,
when chemical modification ofcarboxyl groups was
performed in 100 mMGME (pH 6.2) and 2 mMEDC at
25°C for 12 h The changes of the pI values were almost
saturated within 12 h even by the modification in the
presence of4 mMand 8 mMEDC, implying that a number
ofthe carboxyl groups on the 23 kDa protein are
non-reactive with the chemical modification reagent In spite of
this extended modification with GME, no significant effects
were observed on the binding and reactivating abilities of
the 23 kDa protein Thus, we conclude that the negative
charges on the 23 kDa protein do not contribute to its
interaction with PSII In contrast, 1–5 or 5–10 amino groups
in total ofthe 20 Lys ofthe 23 kDa protein were modified
with NSP when the protein was treated with 0.5 mM or
4 mMNSP at 25°C for 90 min, respectively This indicates that amino groups ofLys residues on the protein are much more reactive than carboxyl groups ofAsp and Glu with respect to the chemical modification reagents
A loss ofthe rebinding ofthe 23 kDa protein following chemical modification can, in principle, be caused by two different mechanisms, as described previously [34] First, chemical modification may induce a conformational change ofthe protein, resulting in a protein structure that is no longer able to bind to the 33 kDa protein associated with the PSII membranes Second, the residues that are modified may participate directly in the electrostatic interaction ofthe
23 kDa protein with the 33 kDa protein associated with the PSII membranes The former possibility appears rather
Table 2 Assignments for peptide fragments from a Staphylococcus V8 protease digest of the extrinsic 23 kDa protein modified with 0.5 mM NSP Deamidation (NG), deamidation ofAsn22–Gly23 to Asp22–Gly23; NP, N-propionyl; Oxydation (M), oxydation ofMet.
Observed mass (Da)
Predicted mass (Da)
Change in mass (%)
Peptide assignment
NSP-modified domains (Lys–Lys)
Deamidation (NG) 855.37 855.40 )0.00 Ser178–Ala186 1107.57 1107.57 1107.57 1107.57 0.00 0.00 0.00 Phe42–Glu50
1222.59 1222.61 1222.60 )0.00 0.00 Phe42–Asp51
1322.72 1322.72 1322.70 1322.73 )0.00 )0.00 )0.00 Gly141–Asp153
1364.71 1364.71 1364.69 1364.71 0.00 0.00 )0.00 Lys40–Glu50
1378.71 1378.72 1378.76 )0.00 )0.00 Gly141–Asp153 + NP 143–152
1402.71 1402.72 1402.76 )0.00 )0.00 Ala5–Glu17
1420.74 1420.73 1420.71 1420.73 0.00 0.00 )0.00 Lys40–Glu50 + NP 40
1479.72 1479.74 )0.00 Lys40–Asp51 1514.81 1514.80 1514.78 1514.81 0.00 )0.00 )0.00 Ala5–Glu17 + 2 NP 11–14 1535.76 1535.76 1535.76 0.00 0.00 Lys40–Asp51 + NP 40
1578.90 1578.91 1578.90 1578.90 0.00 0.00 0.00 Lys166–Glu177 + NP 166–174 1634.93 1634.92 1634.93 0.00 )0.00 Lys166–Glu177 + 2 NP 166–174 1636.92 1636.91 1636.90 1636.76 0.01 0.01 0.01 Ser99–Glu115
1718.86 1718.86 1718.85 1718.85 0.00 0.00 0.00 Tyr86–Glu100
1728.97 1728.97 1728.96 1728.96 0.00 0.00 0.00 Gly25–Glu39
1785.00 1785.00 1784.98 1784.98 0.00 0.00 0.00 Gly25–Glu39 + NP 27–28
2507.22 2507.23 2507.22 2507.22 0.00 0.00 0.00 Asp51–Asp73
2523.22 2523.22 2523.21 2523.22 0.00 0.00 )0.00 Asp79–Glu100
2554.24 2554.25 2554.26 )0.00 )0.00 Phe18–Glu39
Deamidation (NG)
Deamidation (NG);
Oxidation (M)
2610.28 2610.28 2610.27 2610.28 0.00 0.00 )0.00 Phe18–Glu39 + NP
Deamidation (NG)
27–38 2626.29 2626.28 2626.24 2626.28 0.00 0.00 )0.00 Phe18–Glu39 + NP
Deamidation (NG);
Oxidation (M)
27–38
2828.48 2828.45 2828.52 )0.00 )0.00 Gly154–Glu177
Trang 7unlikely on the basis ofthe following considerations Ifloss
ofthe binding ability ofthe 23 kDa protein is caused by
conformational changes following chemical modification,
its binding ability should similarly decrease upon
modifica-tion ofcarboxyl groups with GME, because chemical
modification with GME results in an addition ofa similar
size ofmethyl ester group as that with NSP, as described in
our previous paper [34] The GME modification did not,
however, affect the binding ability of the 23 kDa protein at
all These considerations indicate that the loss ofthe binding
ability ofthe NSP-modified 23 kDa protein is due to
neutralization ofpositively charged lysyl residues ofthe
protein, though conformational changes induced by the
chemical modification cannot be completely excluded
Thus, we conclude that positive charges oflysyl residues
ofthe 23 kDa protein are important for its binding to the
33 kDa protein associated with PSII membranes, whereas negative charges ofcarboxyl groups ofthe 23 kDa protein
do not participate in its binding This conclusion is consistent with the hypothesis predicted by Bricker and Frankel [24] that negative charges on the 33 kDa protein are important for electrostatic interaction with the 23 kDa protein
The locations oflysyl residues on the 23 kDa protein that were modified with NSP were determined in the present study It should be noted again that the modified
23 kDa protein is composed ofproteins having different numbers ofamino residues modified In fact, the band of the modified protein appeared much broader than the unmodified protein upon isoelectric focusing (Fig 1) The
Table 3 Assignments for peptide fragments from a Staphylococcus V8 protease digest of the extrinsic 23 kDa protein modified with 4 mM NSP Deamidation (NG), deamidation ofAsn 22–Gly23 to Asp22–Gly23; Oxydation (M), oxydation ofMet; NP, N-propionyl.
Observed mass (Da)
Predicted mass (Da)
Change in mass (%)
Peptide assignment
NSP-modified domains (Lys–Lys)
Deamidation (NG)
Deamidation (NG);
Oxidation (M) 1107.57 1107.58 1107.57 1107.57 0.00 0.00 0.00 Phe42–Glu50
1222.59 1222.62 1222.60 )0.00 0.00 Phe42–Asp51
1322.71 1322.69 1322.73 )0.00 )0.00 Gly141–Asp153
1364.71 1364.70 1364.70 1364.71 0.00 )0.00 )0.00 Lys40–Glu50
1378.72 1378.76 )0.00 Gly141–Asp153 + NP 143–152 1402.73 1402.69 1402.69 1402.76 )0.00 )0.00 )0.00 Ala5–Glu17
1420.73 1420.73 1420.73 1420.73 0.00 0.00 0.00 Lys40– Glu50 + NP 40
1514.83 1514.80 1514.80 1514.81 0.00 )0.00 )0.00 Ala5–Glu17 + 2 NP 11–14 1535.78 1535.77 1535.76 0.00 0.00 Lys40–Asp51 + NP 40
1634.95 1634.92 1634.94 1634.93 0.00 )0.00 0.00 Lys166–Glu177 + 2 NP 166–174
1690.98 1690.97 1690.96 1690.96 0.00 0.00 0.00 Lys166–Glu177 + 3 NP 166–174 1718.89 1718.87 1718.85 0.00 0.00 Tyr86–Glu100
1728.99 1728.98 1728.96 0.00 0.00 Gly25–Glu39
1774.92 1774.90 1774.88 0.00 0.00 Tyr86–Glu100 + NP 90–96 1834.91 1834.92 1834.86 0.00 0.00 Asp51–Asp67
1841.05 1841.03 1841.03 1841.01 0.00 0.00 0.00 Gly25–Glu39 + 2 NP 27–38
2016.02 2016.06 2016.07 )0.00 )0.00 Ala55–Asp73
2507.24 2507.23 2507.22 0.00 0.00 Asp51–Asp73
2523.24 2523.22 2523.20 2523.22 0.00 0.00 )0.00 Asp79–Glu100
2563.31 2563.23 2563.27 0.00 )0.00 Asp51–Asp73 + NP 68–69 2579.29 2579.25 2579.21 2579.24 0.00 0.00 )0.00 Asp79–Glu100 + NP 90–96
2666.33 2666.27 2666.31 0.00 )0.00 Phe18–Glu39 + 2 NP
Deamidation (NG)
27–38 2682.31 2682.27 2682.30 0.00 )0.00 Phe18–Glu39 + 2 NP
Deamidation (NG);
Oxidation (M)
27–38
2684.27 2684.40 )0.00 Gly141–Asp165 + NP 143–152
Deamidation (NG)
11–14
Trang 8changes ofpI values were estimated to correspond to
modifications of1–5 amino groups in the 0.5 mM
NSP-modified protein However, our mass spectroscoic analysis
indicated that there were eight lysyl residues that were
modified (Results, Fig 4) These facts indicate that lysyl
residues ofthe 23 kDa protein were heterogeneously
modified; some lysyl residues may be modified in a
fraction of the protein by NSP but not in other fractions
ofthe protein The 23 kDa protein modified with 0.5 mM
NSP lost about 82% ofits binding and reactivating
capabilities (Figs 2 and 3, Table 1) In other words, 18%
ofthe binding and reactivating capabilities were still
retained after NSP modification This may well be
attributed to the heterogeneous modification oflysyl
residues NSP modifies not only the lysyl residues required
for electrostatic interaction with the 33 kDa protein but
also the residues not involved in the interaction In
conclusion, we propose that the candidates for
electro-static interaction ofthe 23 kDa protein with the 33 kDa
protein associated with the PSII membranes are at least
present in lysyl residues ofthe six domains ofLys11–
Lys14, Lys27–Lys38, Lys40, Lys90–Lys96, Lys143–
Lys152, Lys166–Lys174 (Fig 4) Complete loss ofthe
binding ability was obtained by treatment with 4 mMNSP
(Figs 2 and 3) in which only one domain, Lys68–Lys69,
was detected to be modified in addition to the six domains
(Fig 4) In the lysyl residues present in the six domains,
11, 13, 33, 38, 143, 166, 170 and 174 (circled K in Fig 4)
were completely conserved in the 23 kDa protein from the
12 species ofhigher plants currently available in databases
The N-terminal region ofthe 23 kDa protein has been reported to be important for its binding with PSII Ifuku and Sato [36] showed that the binding affinity of a recombinant mutant ofthe 23 kDa protein, ofwhich the N-terminal 19 residues were truncated, were apparently weaker than that ofthe native 23 kDa protein Two lysyl residues, Lys11 and Lys13, in the N-terminal 19 residues were modified with NSP and thus these lysyl residues are likely to participate in the electrostatic interaction The negative charges ofGlu1, Glu32, Glu139 and/or Glu187 on the 33 kDa protein have been suggested to be important for the binding of the 23 kDa protein [24] Our current results thus indicate that at least some ofthe positive charges ofthe lysyl residues in the six domains ofthe 23 kDa protein interact electrostatically with these negative charges ofthe 33 kDa protein Some ofthe positive charges on the 23 kDa protein may also be important for its interaction with PSII intrinsic proteins Which residues in these modified lysyl residues directly participate in the electrostatic interaction with the
33 kDa protein (and PSII intrinsic proteins) cannot be identified at present The present study, however, pro-vides important clues for site-directed mutagenesis studies
to identify the lysyl residues that directly participate in the electrostatic interaction
Acknowledgements
We thank ProfJian-Ren Shen ofOkayama University for his critical reading ofthe manuscript.
Fig 4 The amino acid sequence of spinach 23 kDa protein Boxes indicate domains containing lysyl residues modified by NSP at both concen-trations of0.5 m M and 4 m M Dashed box is the domain modified only by 4 m M NSP but not by 0.5 m M NSP The number oflysyl residues modified with 0.5 m M and 4 m M NSP in each domain are shown below each box The circled amino acids indicate lysyl residues that are completely conserved in 12 species ofhigher plants currently available in data bases The sequences were obtained from SwissProt and TrEMBL databases Arrows show the cleavage sites with Staphyrococcus V8 protease.
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